The insulin-like growth factors IGF-I and IGF-II are 7500 dalton proteins chemically related to insulin that stimulate cell survival and proliferation by binding to signaling IGF-I receptors. The IGFs also bind with high affinity to a family of six secreted IGF binding proteins (IGFBPs), forming complexes that are biologically inactive because they cannot bind to IGF-I receptors. In addition, some of the IGFBPs, notably IGFBP-3, can act directly and independently of binding IGFs to stimulate apoptosis and inhibit cell proliferation. During the past year, our ongoing studies of the regulation and biological role of the IGFBPs have focused on the molecular mechanisms by which insulin inhibits IGFBP-1 transcription and the IGF-independent mechanisms by which IGFBP-3 induces apoptosis in human prostate cancer cells. (i) Insulin inhibition of Foxo1-stimulated IGFBP-1 transcription. Insulin inhibits the transcription of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, two genes encoding key enzymes in hepatic gluconeogenesis, and IGFBP-1, which inhibits IGF action and is a critical hepatic survival factor. The three genes share a common insulin response element (IRE) in the proximal promoter. The FOXO subfamily of forkhead transcription factors binds to the IRE to stimulate transcription. Insulin stimulates phosphatidylinositol (PI) 3-kinase-dependent phosphorylation of 3 consensus protein kinase B (PKB)/Akt sites in the FOXO proteins, leading to the export of the transcription factors from the nucleus and to inhibition of FOXO-stimulated transcription. We have examined the mechanisms by which insulin inhibits mouse Foxo1-stimulated IGFBP-1 transcription. First, we evaluated the prevailing hypothesis that nuclear export of the FOXO transcription factor was responsible for insulin inhibition of transcription. We mutated Leu375, a critical residue in the leucine-rich nuclear export signal of Foxo1, to alanine to determine whether IGFBP-1 transcription stimulated by the mutant would still be inhibited by insulin. Immunofluorescence microscopy confirmed that transfected L375A-Foxo1 localized predominantly to the nucleus of H4IIE rat hepatoma cells even after insulin treatment. Despite retention of the Foxo1 mutant in the nucleus, insulin still was able to inhibit L375A-Foxo1-stimulated transcription of two luciferase reporter genes: one controlled by the native IGFBP-1 promoter which contains 2 copies of the IRE, the other by a synthetic promoter which has 3 copies of the IRE. Our results demonstrate that insulin can inhibit Foxo1-stimulated transcription by mechanisms other than nuclear export, for example, direct inhibition of Foxo1 binding to DNA or inhibition of transactivation. To determine whether insulin inhibited transactivation by Foxo1, we fused the C-terminal transactivation domain of Foxo1 (residues 208-652) to the yeast Gal4-DNA binding domain (DBD) and studied activation of a reporter gene in which the Gal4 binding element was fused to luciferase. Gal4DBD-Foxo1 208-652 stimulated Gal4 promoter activity, and transactivation stimulated by the fusion protein was inhibited by insulin. Insulin inhibition of Foxo1 208-652-stimulated transactivation is mediated by PI 3-kinase but, in contrast to full-length Foxo1, does not require the two PKB/Akt phosphorylation sites in the protein fragment. Using mutational and deletion studies, we identified two other potential phosphorylation sites, as well as a 15-amino acid region, that are critical for insulin inhibition of Foxo1 208-652-stimulated transactivation. We conclude that insulin can regulate the transcriptional activity of Foxo1 at the level of transactivation as well as DNA binding and nuclear exclusion. We have begun to investigate the ability of the transcription coactivator/acetyltransferase p300 to regulate Foxo1-stimulated IGFBP-1 transcription. Cotransfection of H4IIE cells with p300 increased Foxo1-stimulated IGFBP-1 promoter activity 12-fold. The intrinsic acetyltransferase activity of p300 was required. Coexpression of p300 also induced acetylation of Foxo1 7-fold. Acetylation was further increased by the deacetylase inhibitor trichostatin A. As with the stimulation of Foxo1-dependent transcription, p300 acetyltransferase activity was required for Foxo1 acetylation, suggesting that acetylation of Foxo1 by p300 may be responsible for the stimulation of transcription. Consistent with this possibility, interaction of Foxo1 and p300 in vitro and in vivo was demonstrated by GST-pulldown and coimmunoprecipitation experiments, and GST-Foxo1 was acetylated by recombinant p300 in vitro. Together these results suggest that direct acetylation of Foxo1 by p300 increases its transcriptional activity. (ii) Characterization of the pathway by which IGFBP-3 induces apoptosis in human prostate cancer cells. We previously reported that an IGFBP-3 mutant (6m-IGFBP-3) that cannot bind IGF-I or IGF-II can induce apoptosis directly in PC-3 prostate carcinoma cells. As a first step in understanding the mechanism by which IGFBP-3 acts, we examined early events in the induction of apoptosis. Apoptosis is the end result of proteolysis of proteins that are necessary to maintain cell integrity by effector or executioner caspases, cysteine-rich proteases that cleave substrates after aspartic residues. The effector caspases first must be activated from inactive precursors by one of two initiator caspase pathways: one involving death receptors / FADD adaptor / caspase 8, the other involving mitochondria / cytochrome c / caspase 9. Tetrapeptide inhibitors of caspase 8 or caspase 9 completely inhibited apoptosis induced by 6m-IGFBP-3, indicating that both pathways were involved. This was confirmed using stable transfectants of PC-3 cells that overexpress dominant negative FADD (FADD-DN) to selectively inhibit the activation of caspase 8, or the antiapoptotic protein Bcl-2 to stabilize mitochondria, preventing the release of cytochrome c and the activation of caspase 9. Caspase 8 activity increased rapidly following addition of 6m-IGFBP-3, reaching a peak at 1 h. The induction of caspase 8 occurred normally in PC-3-Bcl-2 cells, but was greatly decreased in PC-3-FADD-DN, indicating that the induction of caspase 8 was FADD-dependent. These results suggest that 6m-IGFBP-3 triggers apoptosis in PC-3 cells by FADD-dependent activation of caspase 8. To activate effector caspases sufficiently to induce apoptosis, the caspase 8 signal must be amplified by cross-activation of the mitochondrial pathway. In fact, after activation by 6m-IGFBP-3, caspase 8 cleaves the cytosolic protein Bid to truncated tBid which translocates to mitochondria and promotes cytochrome c release. The resulting activation of caspase 9 activates effector caspases to induce apoptosis, thereby amplifying the activation of procaspase 8 in a FADD-independent manner. The molecular mechanisms by which IGFBP-3 triggers FADD aggregation and activation remain to be elucidated.